Project Summary Vibrio cholerae causes the waterborne diarrheal disease, cholera, with annual fatality rates reaching 120,000 worldwide. Environmental survival, transmission, infectivity and dissemination of the pathogen are modulated by its ability to form biofilms, surface-attached microbial communities that are composed of microorganisms and a matrix composed of extra-polymeric substances, such as exopolysaccharides, proteins, and nucleic acids. We seek to understand the molecular mechanism of biofilm formation and its regulation in V. cholerae by studying the production and regulation of the polysaccharide component of the biofilm matrix. In Aim 1, we will determine molecular mechanisms involved in Vibrio polysaccharide (VPS) production and degradation. We will analyze how the tyrosine phosphoregulatory system encoded within the vps biosynthesis gene clusters impacts VPS biosynthesis, and will determine the mechanism and consequences of structural modification of VPS by acetylation. As we have identified a putative VPS lyase, we will determine how this protein impacts VPS production/degradation. In Aim 2, we will determine the mechanism by which two transcriptional regulators, VxrB and CarR, to positively and negatively control vps expression and biofilm formation in V. cholerae. We will also determine the importance of VPS and its regulators for the infection cycle of the pathogen. A better understanding of matrix biosynthesis and regulation will likely allow us to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of Vibrio cholerae.